Dual Immunofluorescence Labeling Protocol
Dual labeling assay using NUNC Labtek II chamberslide system (Fisher Scientific). For this assay, system was HA epitope-tagged (N-terminal) human P2Y2 retrovirally (pLXSN) infected into adherent 1321N1 human astrocytes that were stably maintained in DMEM 5% FBS, penn/strep and 500 ul/ml G418 (selection). Entire assay performed in a sterile tissue culture hood with minimal light-fluorescent or natural.
Procedure
- Grow cells on labtek II slides to subconfluency (70%) at which point wash them and replace with serum free media for overnight (for purinergic receptors only; add 2 units/ml of a-pyrase in ddH20 to the starvation media solution to minimize receptor activiation/internalization due to secreted nucleotide phosphates)
- Prior to starting the first labeling incubation prepare 40 ml 0.5% triton X100 per 2 slides and 40 ml 1% formalin phosphate / 2 slides in beakers or trays. The actual anitbody incubations will take place in 100 x 100 mm square multiuse trays (blotting trays) so you will need to cut out square pieces of filter paper and wet them with ddH20 and place them in the 100 x 100 um trays to maintain a humid environment during the antibody exposure. Prepare 40 ml DMEM +G418 (or whatever media) per two slides and warm to 37 degrees celsius.
- Work with two slides at a time. Prepare one serum free DMEM wash in a 100 ml beaker. The first anitbody incubation is high affinity anti-HA rat monoclonal (12CA5) used at 0.1ug/100 ul. It takes approximately 1 ml of antibody solution (DMEM and G418 no serum or antibiotics) to assay 2 slides. Prepare the antibody solution and subsequently remove the wells using the tray removal tool (dump out media first). Work swiftly to prevent cells from drying. Dip the entire slide gently into the media wash. Gently remove and shake to release residual media. Place the slide into the 100 x100 mm tray. (Preheated to 37 degress with dH20 moistened filter paper) and gently add antibody solution. I used 300 ul per 8 well labtec slide applying 150 ul at a time. Incubate in 37 degree culture chamber for sixty minutes.
- Five to ten minutes prior to the end of the first anitbody incubation, prepare your secondary (fluorochrome) anitbody labeling setup. This includes three washes in 100 ml beakers, two PBS washes and one serum free media wash and a clean 100 x 100 mm multiuse tray preheated with new moistened filter paper. Take slides that have been incubated for sixty minutes with HA probe and do the sequential washes one slide at a time. Lift the slide and gently dip and very gently swirl in PBS washes 3-5x, then finaly the DMEM (culture media wash) 3-5x and add the next antibody (in same fashion as the first) which is the fluoresent conjugate directed against HA probe (in this case oregon green 488 (molecualr probes), (1:100). Place the slides in a small light proof box. Incubate this anitbody for sixty minutes at 37 degress in culture chamber.
- Five to ten minutes prior to the end of the second antibody incubation, set up three washes in 100 ml beakers, two PBS washes and one serum free media wash. Wash the slides as in step 4, but now place your slides (you want them to be completely submerged) in DMEM serum free in 100 x100 mm trays, place in light proof box, and allow to equilibrate 60 minutes in culture chamber.
- You are now at the assay step. basically the cells are living at this point and have a flourescent chromophore label on the receptor via the HA probe and have been reequilibrated. To assay P2Y2, I used agonist concentration 100 uM UTP in DMEM serum free. I will assay an entire slide, either negative or positive UTP to keep the assay as controlled as possilbe (while surface tension between well dividers is sufficient to prevent bleeding of drops on the glass there is the potential for bleeding which is not good if you try to assay and + and – on a single slide). After three minutes exposure to 100 uM UTP, arrest the cells by placing the slide in 4 degree ice cold, 1% formalin PBS for ten minutes. After fixation, vacuum out formalin solution and replace with ice cold 0.5% triton x-100 for one minute to permeabilize cells. Subsequently vacuum out triton and add TBS. Prepare 2 TBS washes and the next antibody solution (1:100 in 3% BSA (EGFR sc-03 SantaCruz)). Wash slides and add next anitbody. Place in 100 x 100 mm tray as preformed in steps 3 and 4. This time incubate in this box and place box in a dark area at r.t. for 60 minutes.
- After incubation, wash the slide in three separate 100 ul baths of TBS and add the second flouresenct conjugate, in this case anit-rabbit CY5 conjugate (Jackson Immunoresearch), 1:100. Incubate r.t. for 60 minutes. Note: Immediately, after you begin this final incubation, prepare the antifade reagent for fixing the slides. ***Prolong antifade kit by Molecular Probes is a good kit to use.
- 5-10 minutes prior to the final incubation prepare 3×100 ml TBS washes and one large 500 ml ddH20 wash. Gently wash the slides through TBS and final water rinse and prop at an angle to allow them to partially dry. (You will see liquid evaporate slowly off slide when faced up. Wait for all liquid to evaporate and add preparative.) Use 25 x 25 mm cover slips and apply approximately 5 ul drop (no air bubbles) to each area (8 areas for 8 well plates) Gently drop the coverslip onto the slide and align. Allow it to settle. Place fixed slide in light proof box and place in 4 degree overnight prior to performing the visualization.
- Assay under confocal scope.
The mounting agent is two component, one powder and one liquid. When you add the liquid make sure all the powder is dissolved. The powder tends to be in a chunks on the bottom of the the jar vial. Vortexing may not loosen up this chunk and into its dissolved state. Also let the soultion set for 5 minutes before pipeting to allow air bubbles to rise out of the glycerol. Molecular Probes Prolong Antifade Kit